Spider silk fusion protein structures for binding to an organic target

Abstract

A protein structure capable of selective interaction with an organic target is provided. The protein structure is a polymer comprising as a repeating structural unit a recombinant fusion protein that is capable of selective interaction with the organic target. The fusion protein is comprising the moieties B, REP and CT, and optionally NT. B is a non-spidroin moiety of more than 30 amino acid residues, which provides the capacity of selective interaction with the organic target. REP is a moiety of from 70 to 300 amino acid residues and is derived from the repetitive fragment of a spider silk protein. CT is a moiety of from 70 to 120 amino acid residues and is derived from the C-terminal fragment of a spider silk protein. NT is an optional moiety of from 100 to 160 amino acid residues and is derived from the N-terminal fragment of a spider silk protein. The fusion protein and protein structure thereof is useful as an affinity medium and a cell scaffold material.

The invention claimed is: 
     
       1. A protein structure capable of selective interaction with an organic target selected from the group consisting of the fragment crystallisable (F c ) region of IgG and molecules comprising IgG or derivatives thereof, wherein said protein structure is a polymer comprising as a repeating structural unit a recombinant fusion protein that is capable of selective interaction with the organic target and comprising the moieties B, REP and CT, wherein the polymer is comprising more than 100 fusion protein structural units, and wherein:
 B is a non-spidroin moiety of more than 30 amino acid residues, which provides the capacity of selective interaction with the organic target, wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, staphylococcal protein A and the E, D, A, B and C domains thereof; variants thereof wherein the B moiety is capable of selective interaction with the organic target; 
 the REP and CT moieties provide the capacity of forming a polymer; 
 REP is a moiety of from 70 to 300 amino acid residues, selected from the group consisting of L(AG) n L, L(AG) n AL, L(GA) n L, L(GA) n GL, wherein
 n is an integer from 2 to 10; 
 each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala; 
 each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly; and 
 each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues; and 
 
 CT is a moiety of from 70 to 120 amino acid residues and has at least 50% identity to SEQ ID NO: 9 or at least 80% identity to SEQ ID NO: 7. 
 
     
     
       2. The protein structure according to  claim 1 , wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, the B domain of staphylococcal protein A, and variants thereof wherein the B moiety is capable of selective interaction with the organic target. 
     
     
       3. The protein structure according to  claim 2 , wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, variants thereof wherein the B moiety is capable of selective interaction with the organic target. 
     
     
       4. The protein structure according to  claim 3 , wherein the B moiety is the Z domain of staphylococcal protein A. 
     
     
       5. The protein structure according to  claim 4 , wherein said recombinant fusion protein is selected from the group of proteins defined by the formulas
   B x -REP-B y -CT-B z  and B x -CT-B y -REP-B z , 
 
       wherein x, y and z are integers from 0 to 5; 
       and x+y+z≧1. 
     
     
       6. The protein structure according to  claim 5 , wherein said recombinant fusion protein is selected from the group of proteins defined by the formulas B x -REP-CT, B x -CT-REP, REP-CT-B z  and CT-REP-B z ; 
       wherein x and z are integers from 1 to 5. 
     
     
       7. The protein structure according to  claim 6 , wherein said recombinant fusion protein is selected from the group of proteins defined by the formulas B-REP-CT, B-CT-REP, REP-CT-B and CT-REP-B. 
     
     
       8. The protein structure according to  claim 1 , wherein the B moiety has less than 30% identity to any of SEQ ID NOS: 6-10. 
     
     
       9. The protein structure according to  claim 1 , wherein said protein structure has a size of at least 0.1 μm in at least two dimensions. 
     
     
       10. The protein structure according to  claim 1 , wherein said protein structure is in a physical form selected from the group consisting of fiber, film, foam, net, mesh, sphere and capsule. 
     
     
       11. The protein structure according to  claim 1 , wherein the CT moiety has at least 50% identity to SEQ ID NO: 9 and at least 80% identity to SEQ ID NO: 7. 
     
     
       12. The protein structure according to  claim 11 , wherein the CT moiety has at least 90% identity to SEQ ID NO: 7. 
     
     
       13. The protein structure according to  claim 12 , wherein the CT moiety is SEQ ID NO: 7. 
     
     
       14. The protein structure according to  claim 1 , wherein the fusion protein is selected from the group consisting of SEQ ID NO: 14 and proteins having at least 80% identity to SEQ ID NO: 14. 
     
     
       15. A method for providing a protein structure capable of selective interaction with an organic target selected from the group consisting of the fragment crystallisable (F c ) region of IgG and molecules comprising IgG or derivatives thereof, wherein said protein structure is a polymer comprising as a repeating structural unit a recombinant fusion protein that is capable of selective interaction with the organic target and comprising the moieties B, REP and CT, wherein the polymer is comprising more than 100 fusion protein structural units, and wherein:
 B is a non-spidroin moiety of more than 30 amino acid residues, which provides the capacity of selective interaction with the organic target, wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, staphylococcal protein A and the E, D, A, B and C domains thereof; and variants thereof wherein the B moiety is capable of selective interaction with the organic target; 
 the REP and CT moieties provide the capacity of forming a polymer; 
 REP is a moiety of from 70 to 300 amino acid residues, selected from the group consisting of L(AG) n L, L(AG) n AL, L(GA) n L, L(GA) n GL, wherein
 n is an integer from 2 to 10; 
 each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala; 
 each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly; and 
 each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues; and 
 
 CT is a moiety of from 70 to 120 amino acid residues and has at least 50% identity to SEQ ID NO: 9 or at least 80% identity to SEQ ID NO: 7, said protein structure displaying a binding activity towards the organic target selected from the group consisting of the fragment crystallisable (F c ) region of IgG and molecules comprising IgG or derivatives thereof, said method comprising the steps of: 
 (a) providing said recombinant fusion protein; and 
 (b) subjecting the fusion protein to conditions to achieve formation of a polymer comprising the recombinant fusion protein. 
 
     
     
       16. An affinity medium for immobilization of an organic target selected from the group consisting of the fragment crystallisable (F c ) region of IgG and molecules comprising IgG or derivatives thereof, said affinity medium comprising a fusion protein that is capable of selective interaction with the organic target and comprising the moieties B, REP and CT, wherein:
 B is a non-spidroin moiety of more than 30 amino acid residues, wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, staphylococcal protein A and the E, D, A, B and C domains thereof; and variants thereof wherein the B moiety is capable of selective interaction with the organic target; 
 the REP and CT moieties provide a capacity of forming a polymer; 
 REP is a moiety of from 70 to 300 amino acid residues, selected from the group consisting of L(AG) n L, L(AG) n AL, L(GA) n L, L(GA) n GL, wherein
 n is an integer from 2 to 10; 
 each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala; 
 each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly; and 
 each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues; and 
 
 CT is a moiety of from 70 to 120 amino acid residues and has at least 50% identity to SEQ ID NO: 9 or at least 80% identity to SEQ ID NO: 7. 
 
     
     
       17. The affinity medium according to  claim 16 , said affinity medium comprising a protein structure capable of selective interaction with the organic target, wherein said protein structure is a polymer comprising as a repeating structural unit the fusion protein that is capable of selective interaction with the organic target and comprising the moieties B, REP and CT, wherein the polymer is comprising more than 100 fusion protein structural units. 
     
     
       18. The affinity medium according to  claim 16 , further comprising said organic target, wherein the B moiety is capable of selective interaction with and is bound to said organic target. 
     
     
       19. The affinity medium according to  claim 18 , wherein said organic target is capable of selective interaction with a second organic target. 
     
     
       20. The affinity medium according to  claim 17 , wherein said protein structure is in the physical form of a film. 
     
     
       21. A cell scaffold material for cultivation of cells having an organic target that is present on the cell surface, said cell scaffold material comprising the protein structure according to  claim 1 , wherein said cell scaffold material is further comprising an intermediate organic target selected from the group consisting of the fragment crystallisable (F c ) region of IgG and molecules comprising IgG or derivatives thereof, wherein the B moiety is capable of selective interaction with and is bound to said intermediate organic target, and wherein said intermediate organic target is capable of selective interaction with the organic target that is present on the cell surface. 
     
     
       22. The cell scaffold material according to  claim 21 , wherein said protein structure is in the physical form of a film. 
     
     
       23. A combination of cells and the cell scaffold material according to  claim 21 . 
     
     
       24. A method for separation of an organic target selected from the group consisting of the fragment crystallisable (F c ) region of IgG and molecules comprising IgG or derivatives thereof from a sample, comprising the steps of:
 providing a sample containing the organic target; 
 providing an affinity medium according to  claim 16 , wherein said affinity medium is capable of selective interaction with the organic target; 
 contacting said affinity medium with said sample under suitable conditions to achieve binding between the affinity medium and the organic target; and 
 removing non-bound sample. 
 
     
     
       25. The method according to  claim 24 , further comprising the step of contacting said affinity medium and the immobilized organic target with a second organic target, which is capable of selective interaction with the first organic target, under suitable conditions to achieve binding between the first and second organic targets. 
     
     
       26. The method according to  claim 24 , wherein the fusion protein in the affinity medium is present as a protein structure when contacting said affinity medium with said sample to achieve binding between the affinity medium and the organic target, said protein structure being capable of selective interaction with an organic target selected from the group consisting of the fragment crystallisable (F c ) region of IgG and molecules comprising IgG or derivatives thereof, wherein said protein structure is a polymer comprising as a repeating structural unit a recombinant fusion protein that is capable of selective interaction with the organic target and comprising the moieties B, REP and CT, wherein the polymer is comprising more than 100 fusion protein structural units, and wherein:
 B is a non-spidroin moiety of more than 30 amino acid residues, which provides the capacity of selective interaction with the organic target, wherein the B moiety is selected from the group consisting of the Z domain of staphylococcal protein A, staphylococcal protein A and the E, D, A, B and C domains thereof; and protein fragments having at least 70% identity to any of these amino acid sequences; 
 the REP and CT moieties provide the capacity of forming a polymer; 
 REP is a moiety of from 70 to 300 amino acid residues, selected from the group consisting of L(AG) n L, L(AG) n AL, L(GA) n L, L(GA) n GL, wherein
 n is an integer from 2 to 10; 
 each individual A segment is an amino acid sequence of from 8 to 18 amino acid residues, wherein from 0 to 3 of the amino acid residues are not Ala, and the remaining amino acid residues are Ala; 
 each individual G segment is an amino acid sequence of from 12 to 30 amino acid residues, wherein at least 40% of the amino acid residues are Gly; and 
 each individual L segment is a linker amino acid sequence of from 0 to 20 amino acid residues; and 
 
 CT is a moiety of from 70 to 120 amino acid residues and has at least 50% identity to SEQ ID NO: 9 or at least 80% identity to SEQ ID NO: 7, when contacting said affinity medium with said sample to achieve binding between the affinity medium and the organic target. 
 
     
     
       27. The method according to  claim 24 , wherein the fusion protein in the affinity medium is present in solution when contacting said affinity medium with said sample to achieve binding between the affinity medium and the organic target, and wherein the complex of fusion protein bound to the organic target is allowed to form the fusion protein structure. 
     
     
       28. The method according to  claim 24 , further comprising the step of detecting, and optionally quantifying, the presence of the immobilized target on said affinity medium. 
     
     
       29. The method according to  claim 24 , further comprising the step of releasing and collecting the organic target from the affinity medium. 
     
     
       30. The method according to  claim 24 , further comprising the final step of regenerating the affinity medium by chemical treatment and/or sterilizing heat treatment. 
     
     
       31. The method according to  claim 30 , wherein the chemical treatment comprises treatment with NaOH and/or urea. 
     
     
       32. A method for immobilization of cells, comprising
 providing a sample comprising cells of interest; 
 applying said sample to the cell scaffold material according to  claim 21 , wherein said cell scaffold material is capable of selective interaction with an organic target that is present on the cell surface; and 
 allowing said cells to immobilize to said cell scaffold material by binding between the organic target on the cell surface and said cell scaffold material. 
 
     
     
       33. A method for cultivation of cells, comprising
 immobilizing cells of interest to a cell scaffold material according to the method of  claim 32 ; and 
 maintaining said cell scaffold material having cells applied thereto under conditions suitable for cell culture. 
 
     
     
       34. The method according to  claim 24 , wherein said protein structure is in the physical form of a film, a fiber or a foam. 
     
     
       35. A recombinant fusion protein consisting of SEQ ID NO: 14. 
     
     
       36. An isolated polynucleic acid selected from the group consisting of nucleic acids encoding the fusion protein according to  claim 35  and SEQ ID NO: 15. 
     
     
       37. A method of producing a fusion protein, comprising the following steps:
 a) expressing in a suitable host the fusion protein according to  claim 35 ; and 
 b) obtaining a mixture containing the fusion protein, and optionally isolating the fusion protein.